Abstract: Background: CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive. Results: We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. Conclusions: Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure.